Electrically Conductive Ink and Uses Thereof

ABSTRACT

The present disclosure provides an electrode including an electrically conductive ink deposited thereon comprising: a nano-scale conducting material; a binding agent; and an enzyme; wherein said ink is essentially solvent free. In one embodiment, the ink includes at least one of a mediator, a cross-linking agent and a substrate as well. In one further embodiment, the electrode provided herein is used in a battery, fuel cell or sensor.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The research and inventions disclosed herein were funded at least inpart by funds received from received from the U.S. Department of theArmy pursuant to SBIR contract numbers W15P7T-06-C-T203 andW15P7T-09-C-S623.

FIELD OF THE DISCLOSURE

The present disclosure pertains to the field of power generation andmore particularly to electrically conductive inks for use in powergeneration. More specifically, the present disclosure pertains to anelectrically conductive ink capable of operating at elevatedtemperatures. Uses of such electrically conductive inks and devicesincorporating the same are also provided.

BACKGROUND

Electrically conductive inks containing enzymes are known in the art. Inmany cases, these inks use either bio-molecules, such as enzymes, oreven whole living organisms to catalyze oxidation of substrates, such asalcohols and carbohydrates to release electrons and generate electricalenergy. Despite substantial research in the field, the prior art inksare often extremely difficult to reliably produce and are extremelytemperature sensitive. These factors have restricted the use andapplications of such electrically conductive inks. Therefore, the art isin need of improved electrically conductive inks with improvedproperties, such as, but not limited to, ease of manufacture andtemperature stability.

The present disclosure provides a solution to this long-felt need. Thepresent disclosure provides an improved electrically conductive ink withthe surprising electrical conductivity properties at elevatedtemperatures. Such a novel feature not been previously disclosed in theart.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows one embodiment of the ink and the anodic current dependencethereof on increasing glucose concentrations.

FIGS. 2A and 2B show a graph of power density vs. voltage of oneembodiment of the ink used in a fuel cell.

FIGS. 3A and 3B show the activity of the enzyme at increasingtemperatures in free solution and electrical current produced for oneembodiment of the ink used in a fuel cell.

FIGS. 4A and 4B show the power curve of a cell and polarization curvesof cathode and enzymatic anode with one embodiment of the ink incombination with a conductive polymer.

FIGS. 5A and 5B show the power curve of a cell and polarization curvesof cathode and enzymatic anode with one embodiment of the ink incombination with a Tetrathiafulvalene (TTF) mediator.

FIG. 6 shows one embodiment of the ink comprising the Lacasse enzyme foroxygen reduction and polarization curve of such electrode without andwith presence of oxygen.

FIG. 7A shows polarization curves of the enzymatic anode one embodimentof the ink when operated at 55 degrees Celsius; polarization curve forcathode is also shown FIG. 7B shows power curve of such cell that ispower density versus cell voltage.

SUMMARY OF THE DISCLOSURE

In a first aspect, the present disclosure provides an electricallyconductive ink exhibiting superior retention of electrical conductivityand biocatalytic activity of embedded enzymes at elevated temperatures.In one embodiment of this aspect, the electrically conductive inkconsists of, consists essentially of or comprises a nano-scaleconducting material, a binding agent and one or more enzymes, and isessentially solvent free. The electrically conductive ink may furthercomprise additional components, including, but not limited to, amediator and a cross-linking agent. In a second aspect, the presentdisclosure provides an aqueous based electrically conductive inkconsisting of, consisting essentially of or comprising a nano-scaleconducting material, a binding agent and one or more enzymes, whereinthe ink is essentially solvent free. The electrically conductive ink mayfurther comprise additional components, including, but not limited to, amediator and a cross-linking agent. In a particular embodiment, theelectrically conductive ink exhibits superior retention of electricalconductivity and biocatalytic activity at elevated temperatures.

In one embodiment of this aspect, the electrically conductive inkexhibits superior retention of electrical conductivity at elevatedtemperatures.

In a third aspect, the present disclosure provides an electricallyconductive ink, existing in a first form and a second form, wherein thefirst form is a water-soluble aqueous based solution which consists of,consists essentially of or comprises a nano-scale conducting material, abinding agent, an aqueous buffer and one or more enzymes and the secondwater insoluble form is generated from the first form and consists of,consists essentially of or comprises the nano-scale conducting material,the binding agent and the one or more enzymes. The electricallyconductive ink, in either or both of the first or second forms, mayfurther comprise additional components, including, but not limited to, amediator and a cross-linking agent. Furthermore, the electricallyconducting ink may be essentially solvent free. In one embodiment ofthis aspect, the electrically conductive ink exhibits superior retentionof electrical conductivity and biocatalytic activity at elevatedtemperatures and is essentially solvent free.

In a fourth aspect, the present disclosure provides a fuel cellcomprising an electrode material, the electrode material furthercomprising an electrically conductive ink of the first through thirdembodiments deposited on said electrode.

DETAILED DESCRIPTION Composition

In one embodiment, the present disclosure provides an electricallyconductive ink exhibiting superior retention of electrical conductivityand biocatalytic activity at elevated temperatures. In one embodiment ofthis aspect, the electrically conductive ink consists of, consistsessentially of or comprises at least one of a nano-scale conductingmaterial, a binding agent and one or more enzymes, and is essentiallysolvent free. The electrically conductive ink may further compriseadditional components, including, but not limited to, a mediator and across-linking agent.

In another embodiment, the present disclosure provides an aqueous basedelectrically conductive ink consisting of consisting essentially of orcomprising a nano-scale conducting material, a binding agent and one ormore enzymes, wherein the ink is essentially solvent free. Theelectrically conductive ink may further comprise additional components,including, but not limited to, a mediator and a cross-linking agent. Ina particular embodiment, the electrically conductive ink the exhibitssuperior retention of electrical conductivity and biocatalytic activityat elevated temperatures.

In still a further embodiment, the present disclosure provides anelectrically conductive ink, existing in a first form and a second form,wherein the first form is a water-soluble aqueous based solutionconsists of consists essentially of or comprises a nano-scale conductingmaterial, a binding agent, an aqueous buffer and one or more enzymes andthe second water insoluble form is generated from the first form andconsists of, consists essentially of or comprises the nano-scaleconducting material, the binding agent and the one or more enzymes. Theelectrically conductive ink, in either or both of the first or secondforms, may further comprise additional components, including, but notlimited to, a mediator and a cross-linking agent. Furthermore, theelectrically conducting ink may be essentially solvent free. In aspecific embodiment, the electrically conductive ink exhibits superiorretention of electrical conductivity and biocatalytic activity atelevated temperatures and is essentially solvent free.

In a specific embodiment, the electrically conductive inks describedabove incorporate a cross-linking agent. In a further specificembodiment, the electrically conductive inks described above incorporatea mediator. In still a further embodiment, the electrically conductiveinks described above incorporate a cross-linking agent and a mediator.

In embodiments of the foregoing where the mediator is not present in theelectrically conductive ink, the mediator may be present in a fluidsolution surrounding the electrically conductive ink. In one embodiment,the mediator is present in a fluid solution surrounding the electricallyconductive ink.

In this embodiment, the first form of the electrically conductive ink ofthe present disclosure is characterized as a water-soluble aqueous basedsolution consisting of consisting essentially of or comprising: anano-scale conducting material, a binding agent, an aqueous buffer andone or more enzymes. The first form may further comprise a mediator anda cross-linking agent. The second form of the electrically conductiveink is characterized as a water insoluble matrix consisting ofconsisting essentially of or comprising the nano-scale conductingmaterial, the binding agent and the one or more enzymes. In thisembodiment, the second form may be generated from the first form bycuring the ink for a period of time. Other methods known in the art mayalso be used. In one embodiment, the second form is generated from thefirst form by self curing the electrically conductive ink on aconductive electrode surface at about 4 degrees Celsius. The first formand or the second form may further comprise a mediator and across-linking agent

In the embodiments of the electrically conductive ink described herein,the electrically conductive ink exhibits increased retention ofelectrical conductivity and biocatalytic activity at increasedtemperatures when compared to prior art electrically conductive inks.For example, the electrically conductive ink retains significantelectrical conductivity and biocatalytic activity at temperatures above40 degrees Celsius or above 50 degrees Celsius as compared to prior artink. In a particular embodiment, the electrically conductive inks of thepresent disclosure retain 75%, 80%, 85%, 90%, 95%, or greater of theirelectrical conductivity and biocatalytic activity while operating orbeing stored at increased temperatures (such as over 40 and 55 degreesCelsius) as compared to 22 degrees Celsius.

In the embodiments of the electrically conductive ink, including thefirst and second forms thereof, described herein the electricallyconductive ink incorporates a nano-scale conducting material. In oneembodiment, the first form and second form of the electricallyconductive ink comprises a nano-scale conducting material. Thenano-scale conducting material may serve several functions including,but not limited to, providing a large surface area to volume for chargetransfer rations that increase the electrode's total biocatalyticreaction rates, immobilizing and stabilizing the enzyme, andfacilitating efficient electron transfer from the enzyme directly to thecathode or anode, and or from the enzyme to the mediator and frommediator to cathode or anode. In one embodiment, the nano-scaleconducting material is a carbon nanotube (CNT). CNTs are cylinder (ortube) shaped molecules of graphitic carbon that have remarkableelectronic properties and many other unique characteristics. In yet afurther embodiment of the present disclosure, the CNT may either be asingle walled or multi-walled CNT. Single walled CNTs comprise a singlerolled layer of graphite carbon while multi-walled CNTs comprisemultiple rolled layers (or concentric tubes) of graphite. In yet afurther embodiment, the single- or multi-walled CNT may befunctionalized. Functionalized CNTs have additional chemical moleculesor functional groups attached to their sidewalls and display increasedsolubility in aqueous liquids and polymer resins as compared tonon-functionalized CNTs. In one further embodiment, the CNT has beenfunctionalized by the addition of a carboxylic acid (COOH) group,however functionalization with other groups including without limitationhydroxyl (OH), amines (NH2), bromine (Br), and others including largerbiomolecules are possible and should be considered within the scope ofthis invention.

In the embodiments of the electrically conductive ink, including thefirst and second forms thereof, described herein the electricallyconductive ink incorporates a binding agent. The binding agent ismiscible or soluble in aqueous solutions. The binding agent serves toimmobilize the nano-scale conductive material, the one or more enzymesand other components of the ink. The binding agent may be any suitableagent as selected by one skilled in the art and may include polymers andother suitable compounds. In one embodiment, the water miscible bindingagent is polymer. In one embodiment, the polymer is a cationic polymer,such as, but not limited to, polyethyleneimine (PEI) polymer.

In the embodiments of the first form of the electrically conductive inkdescribed herein the electrically conductive ink incorporates an aqueousbuffer. In one embodiment, the aqueous buffer is potassium phosphatebuffered saline (PBS) of concentration range of 5 mM to 1M. In alternateembodiments, the aqueous buffer may be(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES),N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic Acid (TAPS),N,N-Bis(2-hydroxyethyl)glycine (Bicine), (hydroxymethyl)aminomethane(TRIS), N-[Tris(hydroxymethyl)methyl]glycine,3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonic acid (Tricine),N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic Acid (TES),3-(N-morpholino)propanesulfonic acid (MOPS),2-[4-(2-sulfoethyl)piperazin-1-yl]ethanesulfonic acid (PIPES),2-(N-morpholino)ethanesulfonic acid (MES) or cacodylate.

In the embodiments of the electrically conductive ink, including thefirst and second forms thereof, described herein the electricallyconductive ink incorporates an enzyme. In certain embodiments, more thanone enzyme may be present. In one embodiment the enzyme may be a singleoxidoreductase enzyme capable of oxidizing or reducing a substrate torelease or consume electrons which may then used to create an electricpotential/current. As is known to those skilled in the art,oxidoreductase enzymes may be oxidases, dehydrogenases or hydrogenases.In one embodiment, the oxidoreductase enzyme is an oxidase which iscapable of oxidizing a carbohydrate substrate. By way of non-limitingexample, in one particular embodiment the oxidase enzyme may glucoseoxidase. In yet a further embodiment, the oxidoreductase enzyme may bedehydrogenase such as pyrrolo-quinoline-quinone (PQQ) glucosedehydrogenase, D-fructose-5-dehydrogenase, glucose dehydrogenase,alcohol dehydrogenase, gluconate 2-dehydrogenase, laccase, bilirubinoxidase, ascorbate oxidase, aldehyde dehydrogenase, oxalate oxidase,malate dehydrogenase, succinate dehydrogenase, pyruvate dehydrogenase,glutamate dehydrogenase, isocitrate dehydrogenase, or lactasedehydrogenase. As will be realized by one skilled in the art, the choiceof one or more enzymes may be influenced by the substrate upon which theone or more enzymes act, the availability of substrate and otherconcerns such as the desired operating environment of the electricallyconductive ink. In one embodiment of the present disclosure, thesubstrate may be a simple or complex carbohydrate, such as, but notlimited to, glucose, fructose, sucrose, trehalose, glycerol, or analcohol, such as, but not limited to, methanol or ethanol. Othersubstrates include ethylene glycol, diethylene glycol, polyethyleneglycol, diol, potentially cellulose, JP8 fuel, methane, butane andother. In a particular embodiment, the enzyme is glucose oxidase.

In yet an alternate embodiment of the present invention, theelectrically conductive ink may contain two or more enzymes. In oneembodiment the two or more enzymes may be part of an enzyme cascade.Enzymes that are capable releasing or consuming a proton/electron inoxidation or reduction reaction respectively generate an electriccurrent/potential. An enzyme cascade might also contain non-electriccurrent/potential enzymes that function as catalysts for chemicalreaction transforming a first substrate or byproduct to a secondsubstance that can be used with the electric current/potentialgenerating enzymes. In one embodiment, the two or more enzymes may bothbe electric current/potential generating enzymes and may be selected sothat the reaction product of one enzymatic reaction may be the substratefor the other enzymatic reaction. As those skilled in the art willrealize, and by way of non-limiting example, the two or more enzymes mayinclude two enzymes selected from the enzymes involved in the Kreb'sCycle (also known as the citric acid cycle) involved in aerobicrespiration or invertase (also known as sucrase) and glucose oxidase forthe hydrolysis of sucrose.

In the embodiments of the electrically conductive ink, including thefirst and second forms thereof, described herein the electricallyconductive ink incorporates a mediator. The mediator may be any compoundthat can assist in the transfer of electrons from the substrate to thenano-scale conductive material and conductive electrode surface or whichcan directly or indirectly increase the efficiency of the oxidation ofthe substrate by the one or more enzymes. In one embodiment the mediatormay be hydroquinone (HQ), ferrocene or ferricyanide. In yet anotherembodiment, the mediator may be an osmium containing compound. In yet analternate embodiment, the mediator may be an azine containing compoundsuch as methyl blue or methyl green. In yet another alternateembodiment, the mediator may be flavin adenine dinucleotide (FAD),nicotinamide adenine dinucleotide (NAD), pyrrolo-quinoline-quinone(PQQ), TTF or osmium based hydrogels. As known to those skilled in theart, the selection of mediator will be influenced by the one or moreenzymes present in the electrically conductive ink and the substrateupon which those enzymes act.

In the embodiments of the electrically conductive ink, including thefirst and second forms thereof, described herein the electricallyconductive ink incorporates a cross-linking agent. The cross-linkingagent functions to assist the transition of the first from of theelectrically conductive into the second from. As known to those skilledin the art, the selection of cross-linker may be dependent upon theselection of the binding agent or one or more enzymes and other factors.By way of non-limiting example, the cross-linker may be1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), aN-hydroxy succinimide (NHS) ester, lysozyme, Dithiobis[succinimidylpropionate (DSP), dithiobis(N-succinimidyl propionate) (DTSP) andpotentially others. In one embodiment, the cross-linking agent is EDC,NHS or combination of the two.

In yet another embodiment, the electrically conductive ink furthercomprises a monomer of an electrically conductive polymer and anelectrolyte. The electrically conductive polymer may be polypyrrole(PPY), polyaniline (PANI), polyacetylene (PAC), poly(p-phenylenevinylene) (PPV) or any other suitable polymer known to those of skill inthe art. A wide selection of electrolytes including ionic liquids mightbe used. The polymerization reaction might be facilitated withelectropolymerization, chemical polymerization, or be also catalyzedwith enzymes. In one preferred embodiment lithium perchlorate (LiClO₄)is the electrolyte. As known to those skilled in the art, the selectionof monomer of an electrically conductive polymer and an electrolyte maybe dependent upon the selection of the one or more enzymes and otherfactors. By way of non-limiting example, the monomer of an electricallyconductive polymer may be electrically polymerized pyrrole or anilinewhile the electrolyte comprises lithium perchlorate.

In a specific embodiment of the foregoing, the electrically conductiveink consists of, consists essentially of or comprises CNTs as thenano-scale conducting material, a binding agent a cross-linking agentand an enzyme. The CNTs may be single walled or multi-walled and may befunctionalized with chemical molecules or functional groups attachedthereto. In one embodiment, the CNT is a single wall CNT with COOHfunctional groups attached thereto. In such an embodiment, the enzyme,binding agent and cross-linking agent may be selected from thosedescribed herein. In one further embodiment of the foregoing, the enzymeis glucose oxidase, the binding agent is PEI and the cross-linking agentis a combination of EDC and NHS. Furthermore, the ink may be essentiallysolvent free.

Uses

The electrically conductive ink of the present disclosure has many uses.For example, the electrically conductive ink may be used as part of anelectrode. The electrode may be used in a diverse range of applications,such as, for example, the construction of a fuel cell, a batteryelectrode or a sensor electrode. The fuel cell then may be used forpowering remote monitors, surveillance devices, sensors (such as sensorscapable of detecting chemical and/or biological weapons), in implantablemedical devices or in the field as part of a battery recharging deviceto recharge batteries in crucial operations (such as on the battlefieldby soldiers) when there is no supply electrical power, but a substratesource is available.

The fuel cell in one embodiment of the present disclosure uses theelectrode comprising the electrically conductive ink as at least one ofan anode or a cathode. In one embodiment, the electrode can be made intoa plate-like or layer-like form, and used in a single layer. The fuelcell can include a reaction vessel capable of storing substrate and ananode and a cathode arranged in the reaction vessel, and the fuel cellelectrode according to the present disclosure is used for at least oneof the anode or the cathode. In one embodiment, the fuel cell electrodecomprises a carbon-based electrode paper, such as Toray paper, GlassyCarbon planar gold surface, gold nanostructured surfaces, gold wire,carbon coated wire or carbon microfibers upon which the ink isdeposited. In one embodiment the carbon-based electrode paper comprisesthe anode of the fuel cell which is separated by a sulfonatedtetrafluoroethylene based fluoropolymer-copolymer membrane, such asNafion® (or any such other suitable proton conductive material) from thefuel cell's cathode. In one embodiment, the cathode is an oxygenreduction cathode that can be designed for an air breathing operation.In one embodiment the oxygen reduction reaction in the cathode might becatalyzed by platinum (Pt), other precious metals, or their combination,or by bioelectrocatalysts like laccase, bilirubin oxidase, ascorbateoxidase and other enzymes. The cathode may comprise various chemistriesthat provide reduction reactions in which protons and electrons areconsumed.

Advantages

The electrically conductive ink of the present disclosure has manysurprising characteristics as compared to the known art. First, asdiscussed in Examples 3 and 7 shown by FIGS. 3A, 3B, 7A and 7B, theelectrically conductive ink renders the one or more enzymes and othercomponents of the ink much less sensitive to elevated temperatures (andlong term storage at elevated temperatures) than the native enzyme andother electrically conductive inks of the prior art. This characteristicprovides the ability for the electrically conductive ink, and devicesincorporating the same, to be operable over a wider range of conditionsthan was previously possible. In addition, the electrically conductiveink of the present disclosure and devices incorporating the same can bestored under conditions that would inactivate prior art electricallyconducting inks and devices depending on such inks, greatly simplifyinguse in real-world conditions. As shown by Examples 3 and 7, theelectrically conductive ink retains conductivity and biocatalyticactivity and the ability to produce electrical power after storage at anelevated temperature as compared to native enzyme. Further, a fuel cellcomprising an electrode upon which the electrically conductive ink hasbeen deposited is environmentally friendly as compared to traditionalbatteries and fuel cells. Finally, the theoretical energy density of afuel cell comprising an electrode upon which the electrically conductiveink has been deposited is approximately ten (10) greater thanlithium-ion batteries.

EXAMPLES Example 1

To demonstrate the functionality the electrically conductive ink of thepresent disclosure, the ability of the electrically conductive ink togenerate electrical current was tested. In this example, theelectrically conductive ink comprised single wall COOH functionalizedCNTs as the nanoscale conducting material, PEI as the binding agent, andglucose oxidase as the enzyme. To create the electrically conductiveink, the polymer was dissolved in a phosphate buffer solution bystirring in a ratio of 100-2000 ug/mL for several hours. After thesolution was sufficiently mixed, CNTs were added to the mixture. In thisexample single-walled CNTs with COOH functionalization were utilized.The CNT ratio was between 1-50 mg/mL and the mixing was accomplished viasonication with an ultrasonicator. Next the enzyme and cross-linkingagents are added to the solution. In this example, the cross linkers EDCand NHS were added along with the enzyme glucose oxidase. To constructthe electrode, a small volume of the electrically conductive inksolution was pippetted onto a Toray paper electrode material. Theelectrode was allowed to cure overnight at approximately 4° Celsius.

The following day the Toray paper electrode was rinsed with phosphatebuffer solution and then attached to a standard glassy carbon electrodefor testing in 3-electrode cyclic voltamettry (CV) test. In this testthe electrode comprising the electrically conductive ink serves as theworking electrode, with a Ag/AgCl (3M KCl) reference electrode and aplatinum counter electrode. CV tests were performed from −0.8V to +0.8Vwith a scan rate of 10 mV/s. In order to test for biocatalytic activityof the enzyme immobilized in the ink, CV sweeps were done withincreasing glucose concentrations of 0, 5, 15, 30, 50, 80, and 120 mM ina test solution of 245 mM phosphate buffer solution with 10 mM HQ addedas an electron mediator. FIG. 1 shows anodic current dependence onglucose concentration (i.e., the current increases as the glucoseconcentration is increased). This test demonstrates the ability of theelectrically conductive ink to oxidize glucose and produce electricalcurrent. For reference a blank piece of Toray paper was also testedunder the same conditions and showed no increase in current with addingglucose.

Example 2

In order to demonstrate power generation, the electrode described inExample 1 was combined with a Pt-based oxygen reduction cathode in afuel cell test configuration. In this example, the electricallyconductive ink was prepared using the same procedure as in Example 1 andthen drop casted onto a carbon felt based electrode. This electrode wasloaded into a custom designed polycarbonate based fuel cell testassembly as the anode side of the fuel cell. For the cathode side of thedevice a Pt-based half-MEA consisting of 0.5 mg/cm² Pt on a gasdiffusion electrode hot pressed to a Nafion 117 membrane was used. Thesubstrate (serving as the “fuel”) comprised 50 mM glucose in 245 mMphosphate buffer solution with 10 mM hydroquinone used as a mediator toimprove electron transport. Power generation was tested by placing thefuel cell under constant load and measuring the resultant voltage andcurrent across that load. The load was varied in steps from an opencircuit condition (5 M-omhs) to a high electric loading condition (10ohms). The maximum power was at ˜500 ohms with a voltage of 0.3 V,current of 2.5 mA·cm⁻² and total power of 1 mW·cm⁻² as shown in FIGS. 2Aand 2B.

Example 3

In order to demonstrate the stability of the ink at elevatedtemperatures during storage the ink was stored at elevated temperaturesfor extended periods of time. As a control experiment, the glucoseoxidase enzyme was mixed in aqueous buffer solution and stored attemperatures of −30° C., +22° C., +40° C., and +55° C. A standard enzymeactivity test (Megazyme) was used to measure the activity of the enzymeafter various storage intervals. The results shown in FIG. 3A establishthat the free enzyme lost enzymatic activity after 24 hrs at +40° C. andalmost immediately at +55° C. As a comparison testing was performed onelectrodes prepared with the enzymatic ink process. The electrodes werecreated using the same electrode preparation and fuel cell testprocedure described in Examples 1 and 2. Multiple electrodes wereprepared and tested. Electrodes were then stored at temperatures of −30°C., +22° C., +35° C., +55° C., and +70° C. with three electrodes beingstored at each condition. The electrodes were tested at periodicintervals up to 3 months. The electrical testing consisted ofmeasurement of current density at a constant resistance of 10 Ohms. Theelectrical testing was used in lieu of the standard assay testing as theimmobilized electrode was not compatible with standard assay techniques.However, the ability to generate electrical current is dependent of theenzymatic oxidation of the glucose substrate and is definitive evidencethat the enzymes are still active. The results in FIG. 3B showsignificant current generation and no degradation in performance afterstorage of up to +55° C. The novel formulation of the electricallyconductive ink of the present disclosure provides for stabilization ofthe enzymes contained therein and allows the electrically conductive inkto function at elevated temperatures.

Example 4

As shown in this example, in one embodiment adding a conductive polymerto the ink improves electrical conductivity and also improves mechanicalstability and properties of ink layer. We added into the ink 1-30% byvolume to the following pyrrole solution; 5-100 mM pyrrole with 10-500mM LiClO₄ mixed in 20-300 mM PBS pH7. After deposition and before theanode testing, pyrrole monomer additive in the deposited ink layer iselectrochemically polymerized for 5-50 seconds at +0.2-1.2 V vs.Ag/AgCl. Anodes fabricated with the addition of the conductive polymer(polypyrrole) were assembled into a fuel cell with air-breathing Ptcathode as described in Example 2. Results from this test are shown inFIGS. 4A and 4B showing >800 μW·cm⁻² at current >3 mA·cm⁻².

Example 5

As shown in this example, in one embodiment adding an electron mediatorto the ink provides immobilized mediator. The specific mediator used inthis example was TTF. A solution of TTF and acetone was mixed and theCNTs were dispersed in this solution. The TTF-CNT solution wascentrifuged and decanted several times and then the supernatant solutionwas mixed with binding agent (PEI), enzyme (glucose oxidase), andcross-linkers (EDC and NHS) as before to make an ink solution. The inksolution was deposited on to a Toray Paper based electrode (anode) whichwas combined with Pt air-breathing cathode and FC hardware as describedin Example 2. The incorporation of the TTF allows for the removal of thediffusive HQ mediator used in Example 2 and the fuel consisted strictlyof glucose and buffer solution (50 mM glucose and 245 mM PBS). The testprocedure was as described in Example 2. Results in FIGS. 5A and 5Bclearly showed enzymatic activity and mediated electron transfer withimmobilized mediator in the ink with power density of >500 μW·cm⁻²generated at current >1.5 mA·cm⁻².

Example 6

In one embodiment, the ink solution comprises enzyme lacasse. The ink ofthis example was prepared using the same protocol as described inExample 1 with the only difference being that the enzyme lacasse wassubstituted for glucose oxidase and provided biocatalyzed oxygenreduction. The electrode was then tested with a electrochemicallytechnique of linear sweep voltammetry against platinum counter electrodein 245 mM PBS buffer pH 5.8 that was (1) degassed with nitrogen and (2)oxygen saturated. FIG. 6 indicates differences in the reduction currentwith and without oxygen presence in the buffer solution. It demonstratesthat multiple enzymes are active in the electrode and operates in directelectron transfer. Typical voltage operation range of such cathode is0.4 to 0.5 V vs. Ag/AgCl.

Example 7

This example shows that a battery comprising the ink of the presentdisclosure can be operated at elevated temperatures. A standard fuelcell was constructed using the glucose oxidase/ink modified anode ofExample 1 and a Prussian Blue (PB) based cathode. Nafion membrane wasused to separate the anodic and cathodic chambers. The anodic chamberwas filled with fuel, as described in Example 2. Prior to filling thecell with fuel, both the fuel stock and the empty cell was placed in a+55° C. temperature controlled chamber for 15 minutes to come tooperating temperature. The fuel was degassed with N₂ gas. After 15minutes the cell was filled with fuel and left to continue equalizationin the temperature controlled chamber for another 15 minutes. Alltesting was performed under the +55° C. operating conditions. Standardpower curve and electrode polarization curves were measured on the cellas described in Example 2.

FIGS. 7A and 7B depict the polarization curves for the anode and cathodeand power curve measured at elevated temperature of 55° C. The powercurve in FIG. 7B shows a partial curve with the peak power density of875 μW·cm⁻² reached at 295 mV. This value of the peak power is verycomparable to those achieved at room (+25° C.) temperature, suggestingthat the ink and batteries containing the ink are stable and perform atelevated temperatures than are known in the art.

Although particular embodiments of the present disclosure have beendescribed, it is not intended that such references be construed aslimitations upon the scope of this disclosure except as set forth in theclaims.

1. An electrode comprising: a. an electrically conductive ink depositedon said electrode, said ink comprising: i. a nano-scale conductingmaterial; ii. a binding agent; and iii. an enzyme; wherein said ink isessentially solvent free.
 2. The electrode of claim 1 wherein said inkmaintains at least 75% of its electrical conductivity and biocatalyticactivity at temperatures up to 55 degrees Celsius as compared to 22degrees Celsius.
 3. The electrode of claim 1 further comprising at leastone of a mediator, a cross-linking agent and a substrate.
 4. Theelectrode of claim 1 wherein the ink exists in a water soluble firstform and a water insoluble second form, said first form comprising anano-scale conducting material, a binding agent, an aqueous buffer andan enzyme and said second form comprises a nano-scale conductingmaterial, a binding agent and an enzyme.
 5. The electrode of claim 4wherein said nano-scale conducting material comprises a carbon nanotube.6. The electrode of claim 5 wherein said carbon nanotube is selectedfrom the group consisting of a single-wall carbon nanotube and amulti-wall carbon nanotube.
 7. The electrode of claim 6 wherein saidcarbon nanotube contains a functional group.
 8. The electrode of claim 7wherein said functional group is a carboxylic acid group.
 9. Theelectrode of claim 1 wherein said binding agent is polyethyleneimine.10. The electrode of claim 1 wherein said enzyme is heat stabilized. 11.The electrode of claim 1 wherein said enzyme comprises at least oneoxidoreductase enzyme capable of oxidizing a substrate.
 12. Theelectrode of claim 11 wherein said oxidoreductase is glucose oxidase.13. The electrode of claim 11 wherein said oxidoreductase enzymecomprises a dehydrogenase.
 14. The electrode of claim 3 wherein saidmediator is selected from the group consisting of hydroquinone,ferrocene, ferricyanide, flavin adenine dinucleotide,tetrathiafulvalene, nicotinamide adenine dinucleotide and an azinecompound.
 15. The electrode of claim 3 wherein said mediator comprises acompound containing osmium.
 16. The electrode of claim 3 wherein saidcross linking agent is selected from the group consisting of1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, a N-hydroxysuccinimide ester, lysozyme and combinations thereof.
 17. The electrodeof claim 3 further comprising a monomer of an electrically conductivepolymer and an electrolyte.
 18. The electrode of claim 17 wherein saidelectrically conductive polymer is selected from the group consisting ofpolypyrrole (PPY), polyaniline (PANI), polyacetylene (PAC),polyp-phenylene vinylene) (PPV) which is electrochemically, chemically,or enzymatically polymerized.
 19. The electrode of claim 17 wherein theelectrolyte comprises lithium perchlorate.
 20. A battery, fuel cell orsensor comprising the electrode of claim 1.